Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Förster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival. Display omitted •Invasive tumor borders and cells proximal to vessels show upregulated Rac1 activity•Rac1 inhibition enhances tumor cell vulnerability during fluid-flow shear stress•Live in vivo imaging of Rac1 activity guides optimal disease progression targeting•Rac1 inhibition reduces intratumoral movement and lung metastasis Floerchinger et al. use spatiotemporal imaging of a Rac1-FRET biosensor mouse to dissect the role of Rac1 in mammary carcinoma metastatic cascade. This image-guided inhibition of Rac1 scheduling at primary sites, during fluid-flow induced shear stress and dissemination vulnerabilities, results in a reduction of disease progression and lung metastasis.